A simple correction for cell autofluorescence for multiparameter cell-based analysis of human solid tumors.

Area of Science:

• Biomedical Engineering
• Cell Biology
• Analytical Chemistry

Background:

• Cell autofluorescence complicates accurate fluorescence signal measurement in cytometry.
• Existing correction methods often require specialized instrumentation or data loss.
• Multiparameter laser scanning cytometry (LSC) is crucial for analyzing human solid tumors.

Purpose of the Study:

• To introduce a simple, cell-by-cell autofluorescence correction method for LSC.
• To validate this method using human breast and lung cancer samples.
• To assess the method's performance under varying correlation conditions.

Main Methods:

• Developed a ratio-based correction using mean autofluorescence (Flauto) to mean total cell fluorescence (Fltotal).
• Applied the method to human breast cancer cell lines and primary lung cancer samples using LSC.
• Compared ratio-based correction with a true cell-by-cell correction and used computer simulations.

Main Results:

• Demonstrated a partial correlation between autofluorescence and Her-2/neu/FITC fluorescence in breast cancer cells.
• Showed that the ratio-based correction results align with true cell-by-cell correction.
• Computer simulations indicated robust mean fluorescence estimates, with minor underestimation of variation when correlations are absent.

Conclusions:

• The proposed ratio correction is effective for autofluorescence in LSC when cell autofluorescence and probe fluorescence are correlated.
• In samples lacking correlation, the method provides reliable mean fluorescence but may slightly underestimate variability.
• This technique offers a practical solution for autofluorescence correction in multiparameter LSC without specialized hardware.